This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There has been no change in the scope of this project. The goal remains to place/recruit bone marrow-derived cells to sites of injury and chronic wounds. To better understand the mechanisms involved and additional ways to achieve these goals, mouse experiments have been implemented. There are two main approaches to using bone marrow-derived stem cells to accelerate wound healing. The first approach, recently reported by our group, is to aspirate bone marrow, expand bone marrow-derived stem cell in vitro, and to physically transfer the cells to the wound site using a suitable delivery system. Using mesenchymal stem cells (MSC) delivered in a fibrin spray, we have shown accelerated healing in human chronic wounds and a possible reduction in scarring. However, this method is laborious and may require repeated bone marrow aspiration, especially for large wounds. We are still continuing to explore this approach, and for that purpose have established a dedicated Good Manufacturing Practices (GMP) facility for that purpose. The second, possibly simpler, approach is to mobilize stem cells from the marrow using a systemically injected cytokine;however, little has been reported about the optimal conditions and parameters for this approach. We also don't know how to best direct the mobilized cells to the site of injury, although we have previously shown that fibrin and products released during fibrin polymerization are chemotactic for inflammatory cells. To answer these questions, we have begun murine studies for determining the best approaches and whether accelerated healing can be achieved by these means. The overall scope of our studies remains the same, namely to use bone marrow-derived stem cells to accelerate healing. The following are our specific aims: 1) To determine the optimal conditions for mobilizing bone marrow populations that are functionally effective for wound repair. Using different preparations of GCSF, we will study the dose and parameters that are best able to mobilize sca-1+ and c-kit+ cells, as well as CD150+/CD48- subpopulations. Flow cytometry and colony formation studies will be used to determine stem cell mobilization. Murine back wounds and dorsal tail wounds will be used to determine wound healing after mobilization. 2) To determine the effect of stem cells on the healing of human chronic wounds. A clinical trial in patients with venous ulcers will determine the effectiveness of bone marrow cells on healing, using fibrin or control topically. Immunolabeling of stem cell markers, including those for mesenchymal stem cells (CD34-/CD45-;CD29+/CD90+), will be used in wound biopsy specimens. Healing will be assessed by computerized planimetry for wound edge migration and healing rate, wound size reduction, and complete closure, or specified in the proposal. 3) To characterize and closely correlate the expression of wound edge molecular markers of impaired healing and epithelial migration in response to treatment. Baseline and sequential biopsies from the edges of venous ulcers treated in specific aim 2 will be used to determine the epidermal expression of c-myc, b-catenin, and keratins 6/16 and 17 at the wounds'edges. These measurements are identical to those specified in the original proposal and, when closely correlated with wound size and edge migration, they will help us establish molecular markers involved in impaired healing and whether stem cell treatment may work by affecting the expression and localization of these specific molecular markers. We conclude that there are optimal conditions to achieve the treatment of wounds with bone marrow-derived stem cells.